The biosynthetic dogma of ribosomally synthesized and posttranslationally modified peptides (RiPP) involves enzymatic intermolecular modification of core peptide motifs in precursor peptides. The plant-specific BURP-domain protein family, named after their four founding members, includes autocatalytic peptide cyclases involved in the biosynthesis of side-chain-macrocyclic plant RiPPs. Here we show that AhyBURP, a representative of the founding Unknown Seed Protein-type BURP-domain subfamily, catalyzes intramolecular macrocyclizations of its core peptide during the sequential biosynthesis of monocyclic lyciumin I via glycine-tryptophan crosslinking and bicyclic legumenin via glutamine-tyrosine crosslinking. X-ray crystallography of AhyBURP reveals the BURP-domain fold with two type II copper centers derived from a conserved stapled-disulfide and His motif. We show the macrocyclization of lyciumin-C(sp³)-N-bond formation followed by legumenin-C(sp³)-O-bond formation requires dioxygen and radical involvement based on enzyme assays in anoxic conditions and isotopic labeling. Our study expands enzymatic intramolecular modifications beyond catalytic moiety and chromophore biogenesis to RiPP biosynthesis.

核糖体合成和翻译后修饰肽 (RiPP) 的生物合成法则涉及前体肽中核心肽基序的酶促分子间修饰。植物特异性 BURP 结构域蛋白家族以其四个创始成员命名，包括参与侧链大环植物 RiPP 生物合成的自催化肽环化酶。在这里，我们展示了AhyBURP，作为创始未知种子蛋白型BURP结构域亚家族的代表，在通过甘氨酸-色氨酸交联的单环枸杞素I和通过谷氨酰胺-酪氨酸交联的双环豆豆素的连续生物合成过程中催化其核心肽的分子内大环化。 AhyBURP 的 X 射线晶体学揭示了 BURP 结构域折叠，具有两个源自保守的钉合二硫键和 His 基序的 II 型铜中心。基于缺氧条件下的酶测定和同位素标记，我们证明了枸杞素-C( sp ³ )-N-键形成的大环化以及豆科蛋白-C( sp ³ )-O-键形成需要双氧和自由基的参与。我们的研究将酶促分子内修饰扩展到催化部分和发色团生物发生之外，扩展到 RiPP 生物合成。